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Biology 3 4 > DNA Manipulation (chapter 4) > Flashcards

DNA Manipulation (chapter 4) Flashcards

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1
Q

Polymerase Enzyme

A

An enzyme that builds a polymer
Synthesizes long chains of amino acids
DNA polymerase synthesizes DNA in a 5’ to 3’ ratio using an existing strand as a template

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2
Q

Ligase

A

Completes DNA strand by creating sugar-phosphate bonds between Okazaki fragments

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3
Q

Okazaki Fragment

A

‘Lagging Strand’ (5’) is replicated in short segments by DNA Polymerase

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4
Q

Endonuclease

A

Enzyme that cuts sugar-phosphate bonds between nucleotides

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5
Q

Palindrome

A

Sequence of double stranded DNA that reads the same on both strands in a 5’ to 3’ direction

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6
Q

Blunt Ends

A

If restriction site is cut directly down the middle

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7
Q

Sticky Ends

A

If cut with overhanging lengths of single stranded DNA, complemented by other ‘sticky ends’ from the same enzyme

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8
Q

Polymerase Chain Reaction

A

A laboratory technique used to produce many identical copies of DNA from a small initial sample (DNA Amplification)

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9
Q

PCR Process

A
  • Identify 25 (or so) sequences at the 3’ end of each strand
  • DNA heated to 95*c, where DNA denatures due to heat
  • DNA primers are created, complementary to 25 bases, made using synthesizers
  • Primers cool DNA to 55*c
  • Reheated to 72c - Taq. polymerase extends primers, because 72 is optimal temperature
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10
Q

Primer

A

Short length of DNA complementary to 3’ end

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11
Q

Taq. Polymerase

A

DNA polymerase taken from bacteria Thermus Aquaticus

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12
Q

CRISPR

A

A naturally occuring adaptive immune response within prokaryotes

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13
Q

Adaptive Immune System

A

System can learn from infection and become more efficient

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14
Q

PAM

A

Protospacer Adjacent Motif (repeating pattern)
a sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9

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15
Q

Spacer

A

DNA captured from bacteriophage viruses that have previously infected the cell

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16
Q

CAS 1 & 2

A

Separate enzymes, work in tandem, look for bases GG (guanine, guanine) and the base before that

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17
Q

GG Base

A

Recognised as PAM

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18
Q

crRNA

A

CRISPR RNA

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19
Q

gRNA

A

Guide RNA

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20
Q

Tracr RNA

A

trans-acting CRISPR RNA

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21
Q

CRISPR Sumamry

A

1- Bacteriophase inserts viral DNA into bactera cell
2- RNA polymerase creates guide RNA (gRNA)
- Transcription occurs
- CRISPR RNA formed
- Endonuclease cuts crRNA and transRNA into targetted gene sequence
3- CAS-9 attaches to gRNA
4- CAS-9 enzyme scanes DNA until it binds to PAM sight (…gg)
5- CAS-9 Binds to PAM site, begins unzipping complementary sequences
6- If gRNA is complementary to DNA, CAS-9 will cut DNA sequence nucleotides from PAM sequence

22
Q

Anneal

A

Combines

23
Q

CAS-9 Structure

A

Protein with a single polypeptide, but six domains

24
Q

sgRNA

A

synthetic guide RNA
used to direct CAS-9 anywhere they want to cut

25
Q

How CAS-9 cuts DNA

A
  • After finding PAM, CAS separates strands, compares base sequences in spacer region of gRNA to bases upstream of PAM, on opposite strand
  • If the sequence is complementary, DNA is cut 4 bases up from PAM
26
Q

Non homologous end Joining

A
  • Joins ends
  • Error-prone, can cause mutation
27
Q

Homology Directed Repair

A

Insert gene into genome to produce gene that wasnt possible before

28
Q

Genome

A

complete set of DNA including genes within an organism
contains info to build and maintain genome

29
Q

DNA technologies

A

Able to isolate and study fragments of DNA
Used to:
- understand evolution
- Investigate disease
- Develop products for medicine

30
Q

Tools for studying and manipulating DNA

A

Naturally occuring enzymes usd by the cell for specific functions
Including:
- Endonucleases
- Ligases
- Polymerases

31
Q

Plasmid

A

Clone short segments of Proteins

32
Q

Gel Electrophoresis

A

A technique that separates DNA fragments based on their molecular size

33
Q

Gel Electrophoresis Process - Preparing the Gel

A
  • Special genetic grade agarose gel melted and poured
  • When gel sets, it forms a pourous structure that DNA can move through - and other fluids
  • Gek tray placed in electrophoresis chamber containing buffer
34
Q

Buffer

A

clear solution, contains ions with electric current

35
Q

Gel Electrophoresis Process - Loading Gel

A
  • Gel (w/ DNA) pipetted into wells at negative electrode of electrophoresis tank
  • Electrophoresis chamber connected to power supply with electrode near DNA
  • Shows where DNA is
  • DNA moves toward positive electrode
  • Smaller strands are able to move further before becoming stuck in matrix
36
Q

STR

A

Short randem respects (short sequence of DNA)

37
Q

Recombinant Plasmids Process

A

1) Choose Restriction Enzyme
2) Choosing Plasmid
3) Using Restriction Endonuclease
4) Making a recombinant plasmid
5) Transforming Bacteria
6) Identifying and Culturing Bacteria

38
Q

Recombinant Plasmid Process - Step 1) Choose Restriction Enzyme

A

Endonuclease is chosen to cut upstream and downstream of gene
Leaves sticky ends
(must cut palindromic region)

39
Q

Recombinant Plasmid Process - Step 2) Choosing a Plasmid

A

Plasmid is chosen that has 2 genes that each encode observable traits
Of these, one must contain the restriction site

40
Q

Recombinant Plasmid Process - Step 3) Using Restriction Endonuclease

A

Same restriction enzyme used to cut both source gene, and the plasmid

41
Q

Recombinant Plasmid Process - Step 4) Making a Recombinant Plasmid

A

When source gene and plasmid are mixed, source gene may incorporate into plasmid, creating a recombinant

42
Q

Plasmid

A

Extra-chromosomal loop of DNA

43
Q

Recombinant

A

Organism or DNA molecule containing DNA from more than one species

44
Q

Recombinant Plasmid Process - Step 5) Transforming Bacteria

A

Bacteria made competent to take up plasmid
- only some bacteria will take up plasmid
- only some will take up recombinant plasmid

45
Q

Recombinant Plasmid Process - Step 6) Identifying and Culturing Bacteria

A

Growing bacteria on agar plate can help determine whether the bacteria has taken up a plasmid
Bacteria that haven’t taken a plasmid wont be able to grow as they lack resistance gene

46
Q

Transgenic Organisms

A

An organism containing a gene from another species

47
Q

Genetically Modified Organism

A

Any organism whose DNA has been modified by gene technology

48
Q

RNAI technology

A

Technique where a gene is silenced by blocking its mRNA

49
Q

Issues with GMO - Animals

A
  • Animal genes in plant products
  • Pig genes in other food - not koscher/halal
  • Animal viruses may affect humans
50
Q

Issues with GMO - Plants

A
  • Concern of spread of GMO - cross pollination
  • New allergens - soybean genes from nuts
51
Q

Issues with GMO - Major Issues

A
  • Monoculture - uniform selective, without variety
  • Loss of genetic diversity - vulnerability
52
Q

What does CRISPR Stand for

A

Clustered regularly interspaced short palindromic repeats

Biology 3 4 (9 decks)

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